The objective of this proposal is to develop and utilize novel chemical probes for activity-based proteomics investigations aimed at understanding the role that hydrolytic enzymes play in tumor metastasis. With the post-genome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been concerned with recording variations in protein level rather than activity. The ability to profile classes of proteins based on changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. The work described in this proposal will focus on designing, synthesizing, and testing novel active site-directed probes that record dynamics in the expression and function of entire enzyme families. In this application, the PIs will use synthetic chemistry, biochemistry, and molecular and cell biology techniques towards the goals of 1) identifying proteases that support or impede tumor metastasis, 2) imaging extracellular "hot spots" of proteolytic activity associated with tumor cell migration and invasion, and 3) developing activity-based probes selective for the matrix metalloproteinase family of enzymes. Through these studies the PI hopes to clarify which, of the many, hydrolytic enzymes implicated in cancer actually participate in proteolytic events important for the progression of the disease. The enzymes will in turn represent valuable targets for pharmaceutical efforts aimed at suppressing cancer metastasis.